Epstein-Barr virus (EBV) is a carcinogenic cofactor for several lymphoid and epithelial cell malignancies (Kieff (2007) Epstein-Barr Virus and its Replication, 5th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia; Rickinson & Kieff (2007) Epstein-Barr Virus, 5th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia; Young & Rickinson (2004) Nat. Rev. Cancer 4:757-68). EBV is associated with the majority of endemic forms of Burkitt's lymphoma and nasopharyngeal carcinomas (NPC). EBV is also found in ˜50% of all Hodgkin's disease tumor biopsies, some forms of gastric carcinoma, thyroid tumors, NK/T cell lymphoma, and the majority of immunosuppression-associated non-Hodgkin's lymphomas and lymphoproliferative disease. Most EBV associated tumors harbor the latent viral genome as a multicopy episome in the nucleus of the transformed cells. During latent infection, EBV does not produce progeny virions, but does express a limited set of viral gene products that promote host-cell survival and proliferation. In proliferating cells, the maintenance of the latent viral genome depends on the functions of the Epstein-Barr Nuclear Antigen 1 (EBNA1) protein (Leight & Sugden (2000) Rev. Med. Virol. 10:83-100). EBNA1 is expressed in all types of EBV latent infection found in proliferating cells and tumors. EBNA1 is essential for the immortalization of primary B lymphocytes by EBV infection (Humme, et al. (2003) Proc. Natl. Acad. Sci. USA 100:10989-94), and its inhibition by siRNA depletion or by ectopic expression of dominant negative mutants causes infected cells death (Kennedy, et al. (2003) Proc. Natl. Acad. Sci. USA 100:14269-14274; Yin & Flemington (2006) Virology 346:385-93).
EBNA1 is a candidate for targeting inhibition of EBV latent infection. EBNA1 is consistently expressed in most, if not all, EBV-associated malignancies (Thorley-Lawson & Gross (2004) N. Engl. J. Med. 350:1328-37). EBNA1 is essential for viral genome maintenance and for infected-cell survival (Kennedy, et al. (2003) supra; Yin & Flemington (2006) supra). Most importantly, EBNA1 is a viral-encoded protein that has well-defined biochemical and structural properties. EBNA1 is composed of two major functional domains, a carboxy-terminal DNA binding domain, and an amino-terminal chromosome tethering domain (Leight & Sugden (2000) supra; Wang, et al. (2006) Mol. Cell. Biol. 26:1124-34). The DNA binding domain is essential for interaction with the viral origin of plasmid replication (OriP) (Yates, et al. (1985) Nature 313:812-815). OriP is composed of a series of 30 bp repeats to which EBNA1 binds an 18 bp palindromic-sequence as a homodimer (Ambinder, et al. (1990) J. Virol. 64:2369-79; Rawlins, et al. (1985) Cell 42:859-68). The DNA binding and dimerization interface have been solved by high resolution X-ray crystallography in the apo- and DNA-bound forms (Bochkarev, et al. (1996) Cell 84:791-800; Bochkarev, et al. (1995) Cell 83:39-46). While there are no known cellular homologues of EBNA1, the three dimensional structure of EBNA1 resembles the overall structure of human papillomavirus (HPV) E2 protein, which has an analogous function to EBNA1 at the HPV origin of DNA replication (Bochkarev, at al. (1995) supra). Protein structure prediction programs suggest that EBNA1 and E2 share structural folds similar to the Kaposi's Sarcoma Associated herpesvirus (KSHV) LANA protein, which shares many functional properties with EBNA1, including DNA binding and episome maintenance of KSHV oriP (Garber, et al. (2002) J. Biol. Chem. 277:27401-11). These observations suggest that EBNA1 is a member of a family of viral origin binding proteins that have no apparent orthologue in the human genome, and therefore may represent attractive targets for inhibitors of viral latent replication and persistence.
Identification of small molecules that specifically inhibit protein-DNA binding activity has had some success (Bowser, et al. (2007) Bioorg. Med. Chem. Lett. 17:5652-5; Kiessling, et al. (2006) Chem. Biol. 13:745-51; Mao, et al. (2008) J. Biol. Chem. 283:12819-30; Rishi, at al. (2005) Anal. Biochem. 340:259-71). Because of the cost-inefficient and time-consuming process of conventional drug discovery over the past decade, high throughput virtual screening (HTVS) has emerged as an attractive and complementary approach to traditional HTS. HTVS typically depends on the availability of a high-resolution crystal structure of the protein target as a template for computational screening. Over the years, HTVS has been applied to the successful identifications of biologically active molecules against targets such as HIV-1 protease, thymidylate, influenza hemagglutinin, and parasitic proteases (Siddiquee, et al. (2007) Proc. Natl. Acad. Sci. USA 104:7391-7396; Vangrevelinghe, et al. (2003) J. Med. Chem. 46:2656-2662).